Human Molecular Genetics, 2013, Vol. 22, No. 9 1746–1754 doi:10.1093/hmg/ddt021 Advance Access published on January 28, 2013 Missense mutations in b-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker–Warburg syndrome Karen Buysse1,{, Moniek Riemersma1,2,{, Gareth Powell4,{, Jeroen van Reeuwijk1,{, David Chitayat5,6,{, Tony Roscioli1,7, Erik-Jan Kamsteeg1, Christa van den Elzen1, Ellen van Beusekom1, Susan Blaser8, Riyana Babul-Hirji6, William Halliday5,6, Gavin J. Wright4, Derek L. Stemple4, Yung-Yao Lin4,9, Dirk J. Lefeber2 and Hans van Bokhoven1,3,∗ 1Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, 2Department of Neurology, Department of Laboratory Medicine, Institute for Genetic and Metabolic Disease, 3Department of Cognitive Neurosciences, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, 6525 GA Nijmegen,theNetherlands,4WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,Hinxton,Cambridge CB10 1SA, UK, 5Mount Sinai Hospital, The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, University of Toronto, M5G 1Z5 Toronto, Canada, 6The Hospital for Sick Children, Division of Clinical and Metabolic Genetics, M5G 1X8 Toronto, Canada, 7School of Women’s and Children’s Health, Sydney Children’s Hospital and the University of New South Wales, Sydney, New South Wales, Australia, 8The HospitalforSickChildren,DivisionofNeuroradiology,M5G1X8Toronto,Canadaand9BlizardInstitute,BartsandThe London School of Medicine and Dentistry, Queen Mary University of London, Newark Street, London E1 2AT, UK ReceivedNovember22,2012;RevisedandAcceptedJanuary18,2013 Severalknownorputativeglycosyltransferasesarerequiredforthesynthesisoflaminin-bindingglycanson alpha-dystroglycan (aDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. MutationsintheseglycosyltransferasegenesresultindefectiveaDGglycosylationandreducedligandbind- ing by aDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly re- ferred to as dystroglycanopathies. The most severe clinical form, Walker–Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the b-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the muta- tions. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of aDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced aDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of aDG and im- plicate B3GNT1 as a novel causative gene for WWS. INTRODUCTION dystrophy-dystroglycanopathy syndromes includes a range of clinical phenotypes. Walker–Warburg syndrome (WWS; Dystroglycanopathies are caused by reduced glycosylation of MIM 236670), muscle–eye–brain disease (MEB; MIM alpha-dystroglycan (aDG) (1,2). This group of muscular 253280) and Fukuyama congenital muscular dystrophy ∗Towhomcorrespondenceshouldbeaddressedat:DepartmentofHumanGenetics855,RadboudUniversityNijmegenMedicalCentre,Nijmegen, POBox9101,6500HBNijmegen,theNetherlands.Tel: +31243616696;Fax: +31243668752;Email:[email protected] †Theauthorswishittobeknownthat,intheiropinion,thefirstfiveauthorsshouldberegardedasjointFirstAuthors. # The Author 2013. Published by Oxford University Press. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by-nc/ 3.0/),whichpermitsnon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercial re-use,[email protected] Human Molecular Genetics, 2013, Vol. 22, No. 9 1747 (FCMD; MIM 253800) represent the most severe end of the mutations in b-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) clinical spectrum. These disorders cause muscular dystrophy can give rise to WWS. and severe eye and brain abnormalities resulting in early in- fantile death (3). The mildest variant of the dystroglycanopa- thies is adult-onset limb-girdle muscular dystrophy (LGMD; RESULTS MIM 607155), associated with mutations in the fukutin- Homozygosity mapping and B3GNT1 mutation analysis related protein (FKRP) gene (4). aDG and beta-dystroglycan (bDG) are central components Toidentify causative mutationsforWWS,wepreviouslyper- of the dystrophin–glycoprotein complex (DGC), which forms formed homozygosity mapping in 30 families with idiopathic alinkbetweenthecytoskeletonandthebasallamina.Theper- WWS using the Affymetrix GeneChip Human Mapping SNP ipheral membrane aDG protein is connected to the cytoskel- Array (21). Eight families showed homozygosity at 11q13, eton via non-covalent binding with the transmembrane bDG containing the B3GNT1 gene, which was associated with protein that is linked to intracellular actin. The link with the aDG glycosylation before in a cellular model of prostate basal lamina is formed by the binding of aDG to several cancer(31).Forthisreason,wefollowedacandidategeneap- tissue-specific extracellular matrix (ECM) proteins, including proach andfocused onB3GNT1inourcohort.Inoneofthese laminin, agrin, perlecan, neurexin and pikachurin (5–11). families(WWS-31),thehomozygousregionwasdelimitedby aDG is highly glycosylated with N-glycans, mucin type SNP_A-4215126 at 11q13.1 and SNP_A-2154685 at 11q13.3 O-glycans and O-mannose type glycans (12–14). aDG– (UCSC hg19 database, http://genome.ucsc.edu, last accessed ligand binding requires specific glycosylation of aDG (6) date on 30 January, 2013), representing a 5.24Mb haplotype through O-linked mannosylation of serine or threonine resi- that was shared among the three affected individuals but dif- dues. The proposed ligand-binding glycan occurs on a ferent from that in an unaffected sibling. In this family, we phosphodiester-linked O-mannose residue (15). detected two homozygous missense mutations in the coding ReducedaDG–ligandbindingcausedbyhypoglycosylation sequence of B3GNT1. No B3GNT1 mutations were detected ofaDGhasbeensuggestedtobetheunderlyingcauseforthe in any of the other seven families with homozygosity at dystroglycanopathies (1,2). Mutations in POMT1, POMT2, 11q13.1,norinanyofthe47additionalfamiliesfromourdys- POMGNT1,LARGE,FKTN,FKRP,ISPDandGTDC2,encod- troglycanopathy cohort. Both mutations are absent in 5379 ing known or putative glycosyltransferases, and a mutation in control samples from the NHLBI GO Exome Sequencing thedystroglycangene(DAG)itselfgiverisetodystroglycano- Project (Exome Variant Server, http://evs.gs.washington.edu/ pathies with specific O-glycosylation defects (4,16–23). Fur- EVS, last accessed date on 30 January, 2013) and in 672 thermore, the phenotypes of patients with mutations in genes exomes of our in-house database. involved in producing the sugar precursor dolichol-phosphate B3GNT1isatypeIItransmembraneproteinandbothmuta- mannose (DOLK, DPM3, DPM2 and likely DPM1) are asso- tions are located in the conserved glycosyltransferase domain ciated with dystroglycanopathies with combined N- and (Glyco_transf_49, pfam13896; Fig. 1; Supplementary Mater- O-linked glycosylation defects (24–26). The mannose group ial, Fig. S1). The first mutation, c.1168A.G (M1), is pre- of dolichol-phosphate mannose is used during the first step dicted to lead to a substitution of asparagine by aspartic acid oftheO-mannosylationofaDGbyanO-mannosyltransferase (p.Asn390Asp), while the second mutation, c.1217C.T complexthatisencodedbyPOMT1andPOMT2(27).Protein (M2), replaces alanine by valine (p.Ala406Val). Screening O-linked-mannose b-1,2-N-acetylglucosaminyltransferase 1 of all available family members showed co-segregation with (POMGnT1)isinvolvedinthesecondstepoftheO-mannosy- disease, with all affected members being homozygous and lation. This enzyme adds an N-acetylglucosamine residue to all unaffected individuals being heterozygous for both the the first mannose (18). The exact functions of the proteins mutations (Supplementary Material, Fig. S2). encoded by FKTN, FKRP, ISPD and GTDC2 are still unknown. However, the protein products of these genes Clinical report might play a role in the glycosylation of the phosphorylated O-mannose glycan (15). LARGE has been shown to act as a The index family (WWS-31) without known consanguinity is bifunctional glycosyltransferase that transfers both xylose ofEastIndiandescentwithfoursiblingsdiagnosedwithWWS and glucuronic acid. These glycan modifications allow aDG and three unaffected sibs (Fig. 1E) (clinical details are tobindECMligands(28).Recently,amutationwasidentified described in the Materials and Methods section). Three preg- in the DAG1 gene, which encodes the dystroglycan precursor nancies were terminated and one affected son died at 2 protein that is post-transcriptionally cleaved into aDG and years of age. He presented with hydrocephalus, Dandy– bDG (29). This mutation, identified in a patient with an Walker malformation, retinal dysplasia, severe hypotonia LGMDphenotype,interferedwithpost-translationalmodifica- and seizures. His creatine kinase (CK) level was very high tions involving LARGE (30). (3180units/l). The magnetic resonance imaging (MRI, Mutation analysis of all known dystroglycanopathy genes Fig.2A–D)showedtypicalWWScharacteristicssuchasven- has revealed the underlying genetic aetiology in (cid:3)50% of tricular enlargement, diffuse widening of the gyri and disor- individuals from our cohort of patients with a severe dystro- ganization of the cortical sulci with areas of cobblestone glycanopathy phenotype, suggesting that more genes remain lissencephaly along the posterior aspects of the occipital tobediscovered.Theidentificationofnewgenesisimportant lobes and temporal lobes. Besides, the white matter, brain to increase insights into the nature of the unknown ligand- stem and cerebellum were clearly affected. From one of the binding glycan. This study provides the first evidence that fetuses, a muscle biopsy was taken. The skeletal muscle 1748 Human Molecular Genetics, 2013, Vol. 22, No. 9 Figure1.SchematicrepresentationofB3GNT1chromosomalposition,proteinstructureandlocalizationofmutations.(A)Ideogramofchromosome11showing thelocalizationofthe SNPsflankingtheshared homozygous regioninthepatients. (B) Zoom-inof the5.2Mb homozygous region.(C) Gene structure of B3GNT1 showing the 5′ UTR, two coding exons separated by the intron and the 3′ UTR. (D) Protein structure of B3GNT1 showing the topological domains, the conserved glycosyltransferase domain and the position of the missense mutations M1 (p.N390D) and M2 (p.A406V). (E) Pedigree of family WWS-31. Individuals that were available for study are identified by their lab number. The mutation status is indicated below each individual (+,present, 2,absent,NA,notavailable). showed a lack of merosin and a-sarcoglycan expression. In glycosylated aDG (1). The number of IIH6-positive cells addition, aDG was not able to bind laminin as assessed by strongly increased on transfection with wild-type B3GNT1 laminin overlay in skeletal muscle homogenate (Fig. 2E). whencomparedwithtransfectionwithanemptyvector.Trans- fection with single mutants and the double mutant did not cause an increase of IIH6-positive cells. Normalization of the results as percentage of IIH6-positive cells in relation to Overexpression of wild-type and mutant B3GNT1 theempty vectorcontrolshowed astatisticallysignificantdif- in human PC3 cells ference in aDG glycosylation between wild-type and mutant To investigate the functional consequences of the mutations, constructs (Fig. 4; P¼0.042). These results indicate that the we first determined the subcellular localization of wild-type identified mutations impair the glycosyltransferase function and mutant B3GNT1. We used human prostate cancer (PC3) of B3GNT1. cells with low levels of endogenous aDG glycosylation (31). We transfected PC3 cells with enhanced green fluorescent protein (EGFP)-tagged wild-type and mutant B3GNT1 con- Morpholino knockdown of zebrafish b3gnt1 structs.Wild-typeandsingleordoublemutantfusionproteins localized to the Golgi apparatus of transfected cells, as deter- Toevaluatethephenotypicconsequencesoflossoffunctionof mined by co-localization with the Golgi marker Giantin B3GNT1 in vivo, we used zebrafish embryos as a model for (GOLGB1, Fig. 3). These results show that the mutations do the dystroglycanopathies (21). The zebrafish ortholog, not affect B3GNT1 subcellular localization. B3gnt1, shows 67% similarity to the human B3GNT1 To investigate the effect of wild-type and mutant forms of protein sequence, including conservation of the two amino B3GNT1 on aDG glycosylation, a flow cytometry assay was acid residues mutated in the family affected by WWS: performed using the IIH6 antibody directed against Asn390 and Ala406 (Supplementary Material, Fig. S1). Human Molecular Genetics, 2013, Vol. 22, No. 9 1749 Figure2.(A–D)MRIat4monthsofage.SagittalT2Wimage(A)reveals hydrocephalus,ahypoplastic‘Z’-shapedbrainstem(arrow)andahypoplastic, dysplasticvermis.CoronalT1Wimage(B)demonstratesabsenceoftheseptal Figure3.Cellularlocalizationofwild-typeandmutantB3GNT1.Wild-type leaflets,verticalhippocampiandfusionofthefornicealcolumnsinthemidline and mutant variants of EGFP-tagged B3GNT1 (green) colocalize with the (arrow).AxialT2Wimage(C)showsfocalcobblestonelissencephalyofthe GolgimarkerGiantin(red).Scalebarrepresents10mm. occipitalcortex(arrow).Thesubjacentwhitematterisabnormallyincreased in signal intensity. A shunt is present in the posterior horn of the right lateralventricle.Inadditiontoventriculomegalyandfocalcobblestonelissen- labels filamentous actin (F-actin) and an antibody against cephaly,coronalT2Wimage(D)revealscysts(arrow)withinthedysplastic bDGtolabelmyotendinousjunctions(MTJs).Musclefibreor- cerebellum. (E) Patient (P) and control (C) muscle homogenates were used ganization and structure were disrupted in morphant embryos foralamininoverlayassay(LO).b-Dystroglycan(b-DG)stainingwasused asloadingcontrol. (Fig. 5D), including muscle fibre detachment and discontinu- ous MTJs, with elongated muscle fibres spanning the myo- septa. Sarcolemma integrity was evaluated by injection of RT-PCR analysis showed that b3gnt1 is expressed in wild- Evan’s blue dye (EBD), which only penetrates the cell when type embryos throughout the first five days of development the membrane is compromised (Fig. 5E). Accumulation of (Fig. 5A). To knockdown b3gnt1, we injected zebrafish EBD was observed in the muscle lesions, indicating muscle embryos with a morpholino designed to disrupt splicing of degeneration with a loss of sarcolemma integrity in b3gnt1 the only intron in the b3gnt1 gene (Fig. 1C). We observed a morphant embryos. great reduction in the expression of the full-length transcript Taken together, these results demonstrate that the missense and the appearance of aberrantly spliced transcripts by mutationsinB3GNT1inthisWWSfamilysignificantlyimpair RT-PCR (Fig. 5B), using complementary DNA (cDNA) its function in vitro as well as in vivo in zebrafish, showing a extractedfrom48hpostfertilization(hpf)morphantembryos. muscle phenotype comparable with dystroglycanopathy. ToassesstheeffectoflossoffunctionofB3gnt1onglyco- sylation of aDG, we extracted cell surface proteins from 48hpf uninjected (positive control), b3gnt1 morpholino- DISCUSSION treated and dag1 morpholino-treated (negative control) embryos. We tested the protein extracts for the presence of Dystroglycanopathies are caused by mutations in (putative) laminin-binding glyco-epitopes by western blot, using the glycosyltransferases and sugar donors that result in aberrant IIH6 antibody (Fig. 5C). Little or no glycosylated aDG was glycosylation of aDG. Identification of all genes involved is observed in extracts from b3gnt1 morphants compared to essential for understanding the pathology in this group of dis- wild-type. These results demonstrate that loss of function of orderswithabnormalglycosylationoftheaDGglycan.Inthis B3gnt1 results in hypoglycosylation (Fig. 5C) and verifies study, we identified two missense mutations in B3GNT1 in a the efficacy of the morpholino. family affected with WWS and showed that these mutations To investigate the effect of b3gnt1 morpholino knockdown are causative for the disease. First, the mutations reside in on the muscle fibre structure and organization, we stained theconservedglycosyltransferasedomain,showcompleteseg- 48hpf morphant and control embryos with phalloidin, which regationwiththediseaseintheindexfamilyandareabsentin 1750 Human Molecular Genetics, 2013, Vol. 22, No. 9 and brain (33). Previous studies in a prostate cancer cell line (28) indicated a role of B3GNT1 in the synthesis of the laminin-bindingglycan.B3GNT1wasoriginallycharacterized asanenzymeinvolvedintheformationofpoly-N-acetyllacto- samine glycans by adding N-acetylglucosamine residues to N-acetyllactosamines attached to N-glycans (33). It has been proposed that B3GNT1 forms a complex with LARGE and that terminal N-acetylglucosamine residues are targets for LARGE glycosyltransferase activity (31,34). One possibility is that B3GNT1 adds a terminal N-acetylglucosamine residue to the phosphodiester-linked glycan that acts as an acceptor for LARGE activity. A recent study has shown that LARGE transfers both xylose and glucuronic acid residues to the unknown ligand-binding glycan (28), perhaps using the N-acetylglucosamine residue transferred by B3GNT1 as initi- ating sugar. It is not yet known how these xylose and glucur- onic acid structures contribute to ligand binding. Together with previous studies, our data suggest that at least three N-acetylglucosaminyl transferases with different specificities are required for synthesis of the ligand-binding glycan on aDG. POMGnT1 is responsible for addition of an N-acetylglucosamine residue in b-1,2 linkage to the first mannose residue. However, the N-acetylglucosamine residue in the phosphodiester-linked O-mannose trisaccharide was proposed in the b-1,4 linkage, while B3GNT1 is supposed to add an N-acetylglucosamine residue in the b-1,3 linkage, likely in the post-phosphoryl glycan (15,33). Altogether, the Figure4.FlowcytometryanalysisoftransfectedPC3cells.PC3cellstrans- synthesis of the laminin-binding glycan on aDG still fected with an empty vector are used as control (A). In B3GNT1 WT remains unclear, necessitating further mechanistic studies to transfectedPC3cells(B)thepercentageofIIH6-positivecellsissignificantly higherthaninPC3cellstransfectedwithanemptyvector(A,F).Thepercen- position uncharacterized proteins as FKRP, FKTN, GTDC2 tages of IIH6-positive cells in B3GNT1 M1 (C), B3GNT1 M2 (D) and and ISPD in the pathway (15,21). B3GNT1M1M2(E)transfectedPC3cellsarecomparablewiththepercentage In conclusion, we have detected two pathogenic missense ofthePC3cellstransfectedwiththeemptyvector(A,summaryinF),indicat- mutations in B3GNT1 which result in impaired glycosylation ingthatglycosylationisaffected.(F)Barchartshowingtherelativeamountof IIH6-positive cells, taking the empty vector control as standard of aDG, giving rise to WWS. Our genetic and functional (n¼3,∗P,0.05,onesampleT-test).Errorbarsshowthestandarddeviation. data provide evidence that B3GNT1 is a novel causative gene for the dystroglycanopathies and recommend its inclu- sion in the diagnostic workup of patients. control cohorts. Second, B3GNT1 overexpression in human PC3 cells results in a significant increase in aDG glycosyla- tion, whereas overexpression of singly or doubly mutated MATERIALS AND METHODS B3GNT1iscomparablewiththenegativecontrol.Thisdemon- Clinical report stratestheinvolvementofB3GNT1inaDGglycosylationand the pathogenicity of the missense mutations. Third, morpho- The index family (WWS-31) is a non-consanguineous family lino knockdown of the zebrafish ortholog b3gnt1 results in of East Indian descent with four affected children and three phenotypic features that are reminiscent of WWS, with unaffectedsiblings(Fig.1E).Themotherhadahistoryofges- muscle structure disorganization being the most prominent tational diabetes. The remainder of the family history is non- finding. Taken together, these results indicate that B3GNT1 contributoryforadditionalriskfactors.Thecouple’sfirstpreg- is required for the interaction between aDG and laminin, as nancy, when the parents were 25 years old, resulted in a impaired B3GNT1 function leads to diminished glycosylation daughter who is well. andsubsequentdisruptionofligandbinding,leadingtopheno- The second pregnancy was complicated with fetal ultra- typic WWS features. Previous genetic analyses in patients sound findings, at 22 weeks of gestation, of hydrocephalus with mild-end dystroglycanopathy phenotypes have not with the lateral ventricles measuring 24mm each and the revealedB3GNT1mutations(32).Furthermore,wehaveiden- third ventricle measuring 5mm. The cerebellum and brain- tifiedonlyonefamilyinourcohortofWWSpatients,suggest- stem were hypoplastic. The pregnancy was terminated at ing that it is a rare cause of dystroglycanopathies. One 24.9 weeks gestation and the autopsy showed diffuse and hypothesis is that more severe mutations might cause embry- severe leptomeningeal neuroepithelial heterotopia, maximal onic lethality and have hitherto remained undetected. over the convexity of the cerebral hemispheres and ventral TheexactfunctionofB3GNT1inaDGO-mannosylationis brainstem. There was obliteration of the subarachnoid space still unknown. B3GNT1 is expressed in tissues typically and diffuse communicating hydrocephalus. There was severe affected in dystroglycanopathies, including skeletal muscle dysplasia/hypoplasia of the cerebellar hemisphere and Human Molecular Genetics, 2013, Vol. 22, No. 9 1751 Figure5.Knockdownofzebrafishb3gnt1causesmuscledefectsandreducedglycosylationofaDG.(AandB)RT-PCRresultsshowingthatb3gnt1isexpressed throughoutzebrafishembryonicdevelopment(A)butgreatlyreducedin48hpfembryostreatedwith6ngor9ngofb3gnt1morpholino(2c,two-cellstage;Shd, shieldstage;d,dayspostfertilization)(B),comparedwithb-actinloadingcontrol;arrowindicatesaberrantlysplicedb3gnt1transcripts.(C)Westernblotusing IIH6antibodytodetecttheaDGglycosylationstate(Glyco.aDag1)in48hpfwild-type(wt),b3gnt1morphant(bMO)ordag1morphant(dMO)embryos. Knockdownofb3gnt1causeshypoglycosylationofaDGcomparedwithwild-type.Ponceaustaining(PonS)loadingcontrolshownbelow.(D)Fluorescentcon- focalmicroscopyimagesof48hpfwild-type(top)andb3gnt1morphant(bottom)embryosstainedwithphalloidin(green),andthecorrespondingDICimages. Lossoffunctionofb3gnt1resultsindisruptedMTJsasindicatedbybDGimmunoreactivity(red)andmusclefibresspanningmultiplesegments.(E)Compro- misedsarcolemmalintegrityprecedesfibredetachmentinb3gnt1morpholino-treatedembryos.Fluorescentconfocalmicroscopyimageofa48hpfembryo, previously injected with b3gnt1 splice-blocking morpholino, treated with EBD (top panel) to highlight muscle fibres with disrupted sarcolemma (arrows). ThecorrespondingDICimage is showninthe middlepanel.Representative imagesof identifiedmuscle lesionsfrom threeindependent experiments; scale barrepresents50mm. vermisandventralbrainstemhypoplasia,maximalatthebasis 14mm, a small cerebellum and agenesis of the corpus callo- pontis. The karyotype was normal (46, XX). sum and inferior vermis. A repeat fetal ultrasound at 21.5 The third pregnancy was complicated with cerebral ventri- weeksgestationshowedhydrocephaluswiththelateralventri- culomegaly involving the lateral and third ventricles with a clesmeasuring17mm.Thecerebellumwasslightlysmalland very thin and smooth cortex at 23 weeks gestation. The cere- there was partial vermian dysgenesis. The couple was coun- bellum was hypoplastic and the cisterna magna was enlarged. selled and decided to terminate the pregnancy. The autopsy Therewasmulticysticdysplasticleftkidneyandveryfewtiny showed afemale fetuswithfindingsconsistentwithWWSin- cysts appeared in the right kidney. The karyotype was normal cluding extensive glio-neuroepithelial leptomeningeal hetero- (46, XY). The couple was counselled and decided to termin- topia with obliteration of the subarachnoid space. There was ate the pregnancy. The autopsy showed a male fetus with a severe communicating hydrocephalus. There was lissence- cystic dysplastic left kidney with a thread-like ureter, testicu- phaly,absentpyramidaltractandagenesisofthecorpuscallo- lar hypoplasia with decreased number and marked size vari- sum. The cerebellum showed severe cortical dysplasia/ ation of seminiferous tubules, 12 ribs on the right and 11 hypoplasia and aplasia of the vermis. ontheleftandX-rayfindingof‘beatensilver’frontalandpar- The couple’s sixth pregnancy resulted in a son, who was ietal skull bones. Neuropathological investigation showed lis- diagnosed prenatally with WWS. The couple was counselled sencephaly type II with cortical dysplasia, severe wavy island and decided to continue the pregnancy. The baby was born architectureandextensiveglio-neuroepithelialleptomeningeal at term via Cesarean section due to severe cerebral ventricu- heterotopiawithobliterationofthesubarachnoidspace.There lomegaly. He presented with hydrocephalus, Dandy–Walker was severe communicating hydrocephalus. The cerebellum malformation, retinal dysplasia, severe hypotonia and intract- showed severe cortical dysplasia/hypoplasia with inferior able seizures. His CK level was very high (3180units/l) and vermian defect. There was hypoplasia of the pyramids at the MRI (Fig. 2) showed ventricular enlargement, diffuse the level of the medulla and the inferior olives had a widening of the gyri and disorganization of the cortical C-shaped dysplasia. There was hydromyelia. The eyes sulci with areas of cobblestone lissencephaly along the pos- showed no anterior segmental abnormalities, focal abnormal- terior aspects of the occipital lobes and temporal lobes. ities involving the retinae of both eyes including the disorga- There was white matter abnormality in association with nized neuronal layer with irregular nests of neurons in the these findings. The brain stem was severely abnormal with nerve fibre layer, some of which appeared to break through wasting of the pons and medulla. The cerebellum was also the inner limiting membrane. There were no abnormalities extremely dysgenetic with cysts, heterotopia and disarray of the extraocular muscles. The findings were consistent of cortical migration. There was absence of the septum pel- with retinal dysplasia. lucidum and fusion of the forniceal columns in the midline. The fourth pregnancy resulted in a son who is well. The globes were apparently intact with thinning of the pos- ThefifthpregnancyresultedinafetuswithWWS.Thefetal terior sclera and retina, consistent with retinal dysplasia. He ultrasound at 17.6 weeks gestation showed a slight ‘lemon’- had a ventriculoperitoneal shunt inserted and died at 2 years shaped head with bilateral ventriculomegaly measuring of age. 1752 Human Molecular Genetics, 2013, Vol. 22, No. 9 Thecouple’sseventhpregnancyresultedinadaughterwho forwardandreverseprimers,5′-TCTTTTTTTTGCTATCCAAAC-3′ is well. and5′-GCATTCATGAGTGTCTCCTTACA-3′.ThefullORF zebrafishb3gnt1cDNAhasbeensubmittedtoGenBank(Acces- sionnumber:KC136354). Patient cohort A cohort of 55 families with one or more individuals affected Western blotting with WWS or MEB were included in this study. Informed consent was obtained from all participants. The study was For human muscle tissues, proteins were extracted from approved by the ethical board of the Radboud University Nij- paraffin-embedded muscle as described (36). Protein samples megen Medical Centre, CMO Regio Arnhem-Nijmegen Ap- were used for western blotting followed by a laminin proval 2011/155. overlay assay and b-dystroglycan staining as described (1,24).Microsomepreparationandwesternblottingusingzeb- rafish embryos were carried out as previously described (21). Homozygosity mapping The primary antibody used in this study was glycosylated Genotyping analysis of genomic DNA was performed using a-dystroglycan IIH6 (Millipore, 1:2000). the Affymetrix GeneChip Human Mapping 10K 2.0 Array or 250K NspI Array. All SNP array experiments were per- Cell culture and transfection formed and analysed according to the manufacturer’s instruc- tions (Affymetrix, Santa Clara, CA, USA). Homozygosity Prostate cancer cells (PC3) (a gift from Gerald Verhaegh of mapping was performed using an in-house algorithm (J.v.R., the Department of Urology, Radboud University Nijmegen unpublished data) for analysis of the genotype files generated Medical Centre) were cultured in RPMI 1690 medium by the Affymetrix GTC software. The number of contiguous (Gibco) supplemented with 10% fetal bovine serum (PAA). homozygous SNPs required for significance in relation to the Cells were transfected using FuGENEw 6 (Roche) according degree of consanguinity for each individual was calculated to manufacturer’s instructions. The ratio of transfection using an algorithm adapted from a previous study (35). reagents (ml) to DNA (mg) used was 6:1. Three days after Regions of excess homozygosity were identified in affected transfection, the cells were used for immunocytochemistry individuals and compared with haplotypes of unaffected or flow cytometry analysis. family members where available. Immunocytochemistry B3GNT1 mutation analysis Transfected and untransfected cells were cultured on glass Sequencing of the two coding exons of B3GNT1 (NCBI Ref- cover slips. The cells were briefly washed using phosphate erenceSequenceNM_006876.2)wasperformedusingtheABI buffered saline (PBS), fixed in 3.7% formaldehyde in PBS at PRISM BigDye Terminator Cycle Sequencing V2.0 Ready room temperature for 10min, permeabilized using 0.4% Reaction kit and analysed with the ABI PRISM 3730 DNA Triton X-100 in 3% BSA/PBS at 48C for 10min, blocked analyzer (Applied Biosystems, Foster City, CA, USA). with3%BSA/PBSatroomtemperaturefor30minandsubse- Primer sequences and PCR conditions are available upon quently incubated with Giantin antibody (Covance) diluted request. 1:400 in 3% BSA/PBS at 48C for 1 hour. Following primary antibody incubation, the cells were briefly washed using PBS and subsequently incubated with Alexa Fluorw 555 Molecular cloning and site-directed mutagenesis Donkey Anti-Rabbit IgG (Molecular Probes) diluted 1:500 Full-length human B3GNT1 mRNA was obtained from in 3% BSA/PBS at 48C for one and a half hour. Cover slips IMAGE cDNA clone 2988041 (Source BioScience). The were embedded in fluorescence mounting medium (DAKO). wild-type human-coding sequences were cloned into the The cells were analysed using a Zeiss Axio Imager Z1 fluor- GatewaypDONRTM201vector(Invitrogen).Site-directedmu- escence microscope (Carl Zeiss). tagenesis using the QuikChangeTM Site-Directed Mutagenesis kit(Stratagene)wascarriedouttointroducethemutationsinto Flow cytometry analysis the constructs. The presence of the mutations was verified by Sanger sequencing. The human c.1168A.G mutation is re- Cells were washed using PBS and subsequently scraped in ferred toas mutation 1(M1).Thec.1217C.Tmutation isre- cold PBS. The cells were blocked using 20% goat serum in ferred to as mutation 2 (M2). Both single (M1 or M2) and 1% BSA/PBS on ice for 20min. The cells were incubated double mutant (M1M2) constructs were designed. Wild-type with IIH6 antibody (Millipore) 1:25 diluted in 1% BSA/PBS and mutant sequences were subsequently cloned into pCS2+ on ice overnight. The cells were washed and subsequently based expression vectors that were used for mRNA synthesis incubated withAlexa Fluorw 647 Goat Anti-Mouse IgG (Mo- and transfection. lecular Probes) 1:200 diluted in 1% BSA/PBS on ice for 2 hours.Thefluorescentsignalofsecondaryantibodywasmea- sured using a CyAn flow cytometer (Beckman-Coulter) with Accession number 642nmlaser.Atotalof75000cellswereanalysedperexperi- To clone full open reading frame (ORF) zebrafish b3gnt1, we ment. Data were analysed using Summit 4.3 software. The carried out RT-PCR using cDNA from 48hpf embryos with percentage of IIH6-positive cells transfected with wild-type Human Molecular Genetics, 2013, Vol. 22, No. 9 1753 and mutant B3GNT1 constructs was normalized against the Deletionofbraindystroglycanrecapitulatesaspectsofcongenital percentage of IIH6-positive cells transfected with the empty musculardystrophy.Nature,418,422–425. 3. vanReeuwijk,J.,Brunner,H.G.andvanBokhoven,H.(2005) vector (Fig. 4F). Statistical significance was determined Glyc-O-geneticsofWalker–Warburgsyndrome.Clin.Genet.,67, usingone-samplet-test(n¼3).AP-valueof ,0.05wascon- 281–289. sidered statistically significant. 4. Brockington,M.,Blake,D.J.,Prandini,P.,Brown,S.C.,Torelli,S., Benson,M.A.,Ponting,C.P.,Estournet,B.,Romero,N.B.,Mercuri,E. etal.(2001)Mutationsinthefukutin-relatedproteingene(FKRP)causea Morpholino and EBD injections in zebrafish formofcongenitalmusculardystrophywithsecondarylaminina2 deficiencyandabnormalglycosylationofa-dystroglycan.Am.J.Hum. Antisense morpholino oligonucleotides (MOs) were obtained Genet.,69,1198–1209. from GeneTools. dag1 MO has been described (37). b3gnt1 5. Henry,M.D.andCampbell,K.P.(1996)Dystroglycan:anextracellular MO (5′-CCTATTCTCCATGTGCTCACCTGGC-3′) was matrixreceptorlinkedtothecytoskeleton.Curr.Opin.CellBiol.,8, designed to target the b3gnt1 exon–intron splice site. All 625–631. 6. Ervasti,J.andCampbell,K.(1993)Aroleforthedystrophin-glycoprotein MOswereinjectedintotheyolkflowatone-cellstageusinga complexasatransmembranelinkerbetweenlamininandactin.J.Cell specified dose in the figure legend. As described (38), 0.1% Biol.,122,809–823. EBD (Sigma) was injected into zebrafish blood circulation at 7. Campanelli,J.T.,Roberds,S.L.,Campbell,K.P.andScheller,R.H.(1994) 48hpf. MO or EBD-injected embryos were fixed using 4% Arolefordystrophin-associatedglycoproteinsandutrophinin PFAforimmunohistochemistryoranalysedliveunderconfocal agrin-inducedAChRclustering.Cell,77,663–674. 8. Gee,S.H.,Montanaro,F.,Lindenbaum,M.H.andCarbonetto,S.(1994) anddifferentialinterferencecontrast(DIC)microscopy. Dystroglycan-a,adystrophin-associatedglycoprotein,isafunctional agrinreceptor.Cell,77,675–686. 9. Peng,H.B.,Ali,A.A.,Daggett,D.F.,Rauvala,H.,Hassell,J.R.and Zebrafish immunohistochemistry Smalheiser,N.R.(1998)Therelationshipbetweenperlecanand Immunostaining of fixed zebrafish embryos was performed as dystroglycananditsimplicationintheformationoftheneuromuscular junction.CellAdhes.Commun.,5,475–489. described (21). Alexa Fluor-conjugated phalloidin (Molecular 10. Sugita,S.,Saito,F.,Tang,J.,Satz,J.,Campbell,K.andSu¨dhof,T.C. Probes; 1:100 dilution) and primary antibody anti-b-Dag1 (2001)Astoichiometriccomplexofneurexinsanddystroglycaninbrain. (Novocastra, 1:50) were used. Alexa Fluorw 488 or 594 con- J.CellBiol.,154,435–446. jugated anti-mouse IgG (Molecular Probes; 1:250 dilution) 11. Sato,S.,Omori,Y.,Katoh,K.,Kondo,M.,Kanagawa,M.,Miyata,K., were used as a secondary antibody. Funabiki,K.,Koyasu,T.,Kajimura,N.,Miyoshi,T.etal.(2008) Pikachurin,adystroglycanligand,isessentialforphotoreceptorribbon synapseformation.Nat.Neurosci.,11,923–931. 12. Holt,K.H.,Crosbie,R.H.,Venzke,D.P.andCampbell,K.P.(2000) SUPPLEMENTARY MATERIAL Biosynthesisofdystroglycan:processingofaprecursorpropeptide.FEBS Lett.,468,79–83. Supplementary Material is available at HMG online. 13. Chiba,A.,Matsumura,K.,Yamada,H.,Inazu,T.,Shimizu,T.,Kusunoki, S.,Kanazawa,I.,Kobata,A.andEndo,T.(1997)Structuresofsialylated O-linkedoligosaccharidesofbovineperipheralnervea-dystroglycan. ACKNOWLEDGEMENTS J.Biol.Chem.,272,2156–2162. 14. Sasaki,T.,Yamada,H.,Matsumura,K.,Shimizu,T.,Kobata,A.and We would like to thank all patients and their familymembers Endo,T.(1998)DetectionofO-mannosylglycansinrabbitskeletal for participation in this study. We also thank Jo (Huiqing) musclea-dystroglycan.Biochim.Biophys.Acta.,1425,599–606. Zhou for insightful discussions and support. 15. Yoshida-Moriguchi,T.,Yu,L.,Stalnaker,S.H.,Davis,S.,Kunz,S., Madson,M.,Oldstone,M.B.A.,Schachter,H.,Wells,L.andCampbell, Conflict of interest statement. None declared. K.P.(2010)O-Mannosylphosphorylationofalpha-dystroglycanis requiredforlamininbinding.Science,327,88–92. 16. Beltra´n-ValerodeBernabe´,D.,Currier,S.,Steinbrecher,A.,Celli,J.,van Beusekom,E.,vanderZwaag,B.,Kayserili,H.,Merlini,L.,Chitayat,D., FUNDING Dobyns,W.B.etal.(2002)MutationsintheO-mannosyltransferasegene POMT1giverisetothesevereneuronalmigrationdisorderWalker– This work was supported by the EU FP7 Health Programme Warburgsyndrome.Am.J.Hum.Genet.,71,1033–1043. (241995 GENCODYS to H.v.B.); the Prinses Beatrix Fund 17. vanReeuwijk,J.,Janssen,M.,vandenElzen,C.,Beltra´n-Valerode (grant W.OR09-15 to D.L. and H.v.B.); the Hersenstichting Bernabe´,D.,Sabatelli,P.,Merlini,L.,Boon,M.,Scheffer,H., Nederland (grant KS 2009(1)-110 to H.v.B.)]; Wellcome Brockington,M.,Muntoni,F.etal.(2005)POMT2mutationscause Trust (WT 077047/Z/05/Z, WT 077037/Z/05/Z, WT 098051 a-dystroglycanhypoglycosylationandWalker–Warburgsyndrome. J.Med.Genet.,42,907–912. to D.L.S. and G.J.W.); and the European Molecular Biology 18. Yoshida,A.,Kobayashi,K.,Manya,H.,Taniguchi,K.,Kano,H.,Mizuno, Organization (Long-Term Fellowship ALTF 805-2009 to M.,Inazu,T.,Mitsuhashi,H.,Takahashi,S.,Takeuchi,M.etal.(2001) K.B.). Funding to pay the Open Access publication charges Musculardystrophyandneuronalmigrationdisordercausedbymutations forthisarticlewasprovidedbytheWellcomeTrust. inaglycosyltransferase,POMGnT1.Dev.Cell,1,717–724. 19. 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